Vagus nerve stimulation pre-screening test

ABSTRACT

Diagnostic screening tests that can be used to identify if a patient is a good candidates for an implantable vagus nerve stimulation device. One or more analyte, such as a cytokine or inflammatory molecule, can be measured from a blood sample taken prior to implantation of a vagus nerve stimulator to determine the patient&#39;s responsiveness to VNS for treatment of an inflammatory disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/103,873, filed Aug. 14, 2018, titled “VAGUS NERVESTIMULATION PRE-SCREENING TEST,” now U.S. Patent Application PublicationNo. 2019/0046799, which claims priority to U.S. Provisional PatentApplication No. 62/545,284, filed on Aug. 14, 2017, and titled “VAGUSNERVE STIMULATION PRE-SCREENING TEST,” which is herein incorporated byreference in its entirety.

The pre-screening methods and apparatuses described herein may berelated to therapies and apparatuses such as those described in one ormore of: the following pending U.S. patent applications: U.S. patentapplication Ser. No. 14/630,613, filed on Feb. 24, 2015, titled “VAGUSNERVE STIMULATION SCREENING TEST,” now U.S. Patent Publication No.2015/0241447; U.S. patent application Ser. No. 12/620,413, filed on Nov.17, 2009, titled “DEVICES AND METHODS FOR OPTIMIZING ELECTRODE PLACEMENTFOR ANTI-INFLAMATORY STIMULATION,” now U.S. Pat. No. 8,412,338; U.S.patent application Ser. No. 12/874,171, filed on Sep. 1, 2010, titled“PRESCRIPTION PAD FOR TREATMENT OF INFLAMMATORY DISORDERS,” PublicationNo. US-2011-0054569-A1; U.S. patent application Ser. No. 12/917,197,filed on Nov. 1, 2010, titled “MODULATION OF THE CHOLINERGICANTI-INFLAMMATORY PATHWAY TO TREAT PAIN OR ADDICTION,” Publication No.US-2011-0106208-A1; U.S. patent application Ser. No. 12/978,250, filedon Dec. 23, 2010, titled “NEURAL STIMULATION DEVICES AND SYSTEMS FORTREATMENT OF CHRONIC INFLAMMATION,” now U.S. Pat. No. 8,612,002; U.S.patent application Ser. No. 12/797,452, filed on Jun. 9, 2010, titled“NERVE CUFF WITH POCKET FOR LEADLESS STIMULATOR,” now U.S. Pat. No.8,886,339; U.S. patent application Ser. No. 13/467,928, filed on May 9,2012, titled “SINGLE-PULSE ACTIVATION OF THE CHOLINERGICANTI-INFLAMMATORY PATHWAY TO TREAT CHRONIC INFLAMMATION,” now U.S. Pat.No. 8,788,034; and U.S. patent application Ser. No. 13/338,185, filed onDec. 27, 2011, titled “MODULATION OF SIRTUINS BY VAGUS NERVESTIMULATION,” Publication No. US-2013-0079834-A1. Each of these patentapplications is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. The following isa list of publications that are incorporated by reference:

-   Andersson U, Tracey K. Reflex principles of immunological    homeostasis. Annu Rev Immunol 2012; 30:313.-   Bruchfeld A, et al. Whole blood cytokine attenuation by cholinergic    agonists ex vivo and relationship to vagus nerve activity in    rheumatoid arthritis. J Int Med 2010; 268:94.-   Dake M. Chronic cerebrospinal venous insufficiency and multiple    sclerosis: Hostory and background. Techniques Vasc Intervent Radiol    2012; 15:94.-   Ellrich J. Transcutaneous vagus nerve stimulation. Eur Neurological    Rev 2011; 6:254-256.-   Gao X, et al. Investigation of specificity of auricular acupuncture    points in regulation of autonomic function in anesthetized rats.    Autonomic Neurosc 1998; 88:109.-   Huston J, et al. Transcutaneous vagus nerve stimulation reduces    serum high mobility group box 1 levels and improves survival in    murine sepsis. Crit Care Med 2007; 12:2762.-   Koopman F, et al. Pilot study of stimulation of the cholinergic    anti-inflammatory pathway with an implantable vagus nerve    stimulation device in patients with rheumatoid arthritis. Arth Rheum    2012; 64 (10 suppl):S195.-   M. L. Oshinsky, A. L. Murphy, H. Hekierski Jr., M. Cooper, B. J.    Simon, Non-Invasive Vagus Nerve Stimulation as Treatment for    Trigeminal Allodynia, PAIN (2014), doi:    http://dx.doi.org/10.1016/j.pain.2014.02.009.-   Peuker E. The nerve supply of the human auricle. Clin Anat 2002;    15:35.-   Tekdemir I, et al. A clinico-anatomic study of the auricular branch    of the vagus nerve and Arnold's ear-cough reflex. Surg Radiol Anat    1998; 20:253.-   Yu L, et al. Low-level transcutaneous electrical stimulation of the    auricular branch of the vagus nerve: A non-invasive approach to    treat the initial phase of atrial fibrillation. Heart Rhythm 2013;    10: 428.-   Zhao Y, et al. Transcutaneous auricular vagus nerve stimulation    protects endotoxemic rat from lipopolysaccharide-induced    inflammation. Evid Based Complement Alternat Med. 2012; 2012:627023.    doi: 10.1155/2012/627023. Epub 2012 Dec. 29.

FIELD

Embodiments of the invention relate generally to systems and methods forusing vagus nerve stimulation for treatment, and more specifically topre-screening methods and apparatuses for identifying patient that mayrespond to vagus nerve stimulation treatment for the treatment ofinflammatory disorders.

BACKGROUND

The vagus nerve mediates the inflammatory reflex, a mechanism thecentral nervous system utilizes to regulate innate and adaptiveimmunity. The afferent arm of the reflex senses inflammation bothperipherally and in the central nervous system, and down-regulates theinflammation via efferent neural outflow. The efferent arm of thisreflex has been termed the “cholinergic anti-inflammatory pathway”(CAP). The reflex serves as a physiological regulator of inflammation byresponding to environmental injury and pathogens with an appropriatedegree of immune system activation (Andersson, 2012). CAP activation canalso be harnessed to reduce pathological inflammation. Activating theCAP chronically using electrical neurostimulation of the vagus nerve isa feasible means of treating diseases characterized by excessive anddysregulated inflammation.

Although vagus nerve stimulation has been shown to be effective in somepatients, the efficacy of the treatment may vary, particularly at low tomoderate stimulation levels. Responders are those patient's thatresponse strongly to vagus nerve stimulation, particularly for thetreatment of inflammatory disorders, including those described above,and incorporated by reference. However, there are some patients for whomvagus nerve stimulation alone, and particularly electrical vagus nervestimulation, may not be sufficient. These patients may be referred to asnon-responders or low responders. It would be particularly helpful to beable to a priori determine if a particular patient will respond well toa vagus nerve stimulation. Described herein are methods and apparatusesthat may be used to identify, e.g., responders from non-respondersand/or low-responders.

SUMMARY OF THE DISCLOSURE

The present invention relates generally to systems and methods for usingvagus nerve stimulation for treatment, and more specifically toscreening tests for identifying suitable patients for vagus nervestimulation treatment.

In general, described herein are method for screening a patient forresponsiveness to vagus nerve stimulation by challenging an extractedblood sample from the patient (either fresh or frozen). Also descriedherein are alternatively or additional methods for determining if apatient will respond to vagus nerve stimulation by comparing patientlevels of a biomarker before and after vagus nerve stimulation.Surprisingly, techniques for examining extracted blood in the absence ofvagus nerve stimulation may predict how well a patient may respond tovagus nerve stimulation, including in particular, vagus nervestimulation to treat an inflammatory disorder using an implanted vagusnerve stimulator.

For example, described herein are methods of treating a patient havingan inflammatory disorder with vagus nerve stimulation. In somevariations, these methods may be methods of determining if a patientwill respond (e.g., is a responder) or will not respond (e.g., is anon-responder) to vagus nerve stimulation and therefore should or shouldnot have a vagus nerve stimulation device implanted. For example, amethod of treating a patient having an inflammatory disorder with vagusnerve stimulation may include: challenging a sample of blood with afirst concentration of toxin, wherein the sample is taken from thepatient prior to implantation of a vagus nerve stimulator; challenging asecond sample of blood with a second concentration of toxin that is atleast 2 times (e.g., 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×,30×, 50×, 100×, etc.) the first concentration, wherein the second sampleis taken from the patient prior to implantation of the vagus nervestimulator; and implanting the vagus nerve stimulator to treat thepatient for the inflammatory disorder when an amount of an inflammatorycytokine released in response to the second concentration of toxin isgreater than an amount of the inflammatory cytokine released in responseto the first concentration of toxin by a threshold.

Any of these methods may include determining a ratio of the amount ofthe inflammatory cytokine released in response to the secondconcentration of toxin and the amount of the inflammatory cytokinereleased in response to the first concentration, and may compare thisratio to a threshold ratio (e.g., a cutoff threshold). For example, thethreshold ratio for the change in inflammatory cytokine release due toincreasing toxin amount may be, e.g., 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, etc. In some variations the threshold ratio is 60 (e.g., apatient is a good candidate if the ratio is greater than 60).

Any of the methods described herein may include displaying an indicationthat the patient is a responder or a non-responder based on the amountof the inflammatory cytokine released in response to the secondconcentration of toxin compared to the amount of the inflammatorycytokine released in response to the first concentration of toxin(including based on the ratio of the inflammatory cytokine release atdifferent levels of toxin).

The methods described herein may include measuring the amount of theinflammatory cytokine that is released in response to the firstconcentration and measuring the amount of the inflammatory cytokine thatis released in response to the second concentration.

As described in greater detail below, the toxin may be an endotoxin,including Lipopolysaccharide (LPS).

Although a first and a second level of toxin are described, one or moreadditional toxin levels may also be described, such as a third or fourthtoxin challenge. For example, the method may include challenging a thirdsample of blood with a third concentration of toxin that is at least 2times the second concentration, wherein the third sample is taken fromthe patient prior to stimulation of the patient's vagus nerve andwherein implanting the vagus nerve stimulator comprises implanting thevagus nerve stimulator when an amount of an inflammatory cytokinereleased in response to the third concentration of toxin is greater thanan amount of the inflammatory cytokine released in response to the firstand second concentrations of toxin by the threshold.

In some variations, challenging the second sample of blood with thetoxin at the second concentration may comprises challenging with aconcentration that is at least 10 times the first concentration.

In general, the first sample of blood and the second sample of blood maybe portions of a master sample taken from the patient. Thus, the sameblood sample may be challenged with different levels of toxin.

The inflammatory cytokine may be one or more of: tumor necrosis factor(TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12(IL-12), interleukin-18 (IL-18), inflammsome, interferon gamma, andgranulocyte-macrophage colony stimulating factor. In some variations,the inflammatory cytokine may be TNF.

For example, a method of treating a patient having an inflammatorydisorder with vagus nerve stimulation may include: obtaining a sample ofblood taken from the patient prior to implantation of a vagus nervestimulator; challenging the sample of blood with LPS at a firstconcentration of LPS; measuring a level of an inflammatory cytokine thatis released in response to the LPS challenge at the first concentrationof LPS; challenging the sample of blood with LPS at a secondconcentration of LPS that is at least 5 times the first concentration ofLPS; measuring a level of the inflammatory cytokine that is released inresponse to the LPS challenge at the second concentration of LPS;determining a response curve based on the level of the inflammatorycytokine that is released in response to the LPS challenges at the firstconcentration of LPS and the second concentration of LPS; and implantingthe vagus nerve stimulation when the determined response is greater thana cutoff threshold.

The second concentration of LPS may be at least 10 times the firstconcentration of LPS. The method may include challenging the sample ofblood with LPS at a third concentration of LPS that is at least 5 timesthe second concentration of LPS, and wherein the response curve isdetermined based on the level of the inflammatory cytokine that isreleased in response to the LPS challenges at the first concentration ofLPS, the second concentration of LPS, and the third concentration ofLPS. The inflammatory cytokine may be selected from the group consistingof TNF, IL-1, IL-6, IL-12, IL-18, inflammsome, interferon gamma, andgranulocyte-macrophage colony stimulating factor. For example, theinflammatory cytokine nat be TNF. The step of determining the responsecurve may include calculating a slope of the response curve. The methodmay include comprising comparing the determined response curve to thecutoff threshold by comparing a slope of the response curve to thecutoff threshold. Implanting may comprises determining whether a slopeof the response curve is greater than the cutoff threshold.

The methods (and apparatuses for performing them) described herein mayinclude in vitro methods for screening a patient for responsiveness tovagus nerve stimulation to treat an inflammatory disorder. For example,a method may include: challenging a sample of blood with a firstconcentration of toxin, wherein the sample is taken from the patientprior to stimulation of the patient's vagus nerve; challenging a secondsample of blood with a second concentration of toxin that is at least 2times the first concentration, wherein the second sample is taken fromthe patient prior to stimulation of the patient's vagus nerve; andoutputting an indication that the patient is a responder or anon-responder for vagus nerve stimulation based on the amount ofinflammatory cytokine released in response to the second concentrationof toxin compared to the inflammatory cytokine released in response tothe first concentration of toxin.

Any of these methods may include determining if the patient is aresponder for vagus nerve stimulation by determining that a ratio of theamount of the inflammatory cytokine released in response to the secondconcentration of toxin and the amount of the inflammatory cytokinereleased in response to the first concentration of toxin is greater thana threshold ratio. For example, the threshold ratio may be greater than60. Any of these methods may include challenging the sample of bloodwith the toxin at a third concentration that is at least 2 times thesecond concentration and measuring a level of the inflammatory cytokinethat is released in response to the third concentration, whereinoutputting the indication that the patient is a responder for vagusnerve stimulation treatment to treat an inflammatory disorder is basedon the amount of inflammatory cytokine released in response to the thirdconcentration of toxin relative to the response to the first and secondconcentrations of toxin.

Challenging the sample of blood with the toxin at a second concentrationmay comprise challenging with a concentration that is at least 10 timesthe first concentration.

An in vitro method for screening a patient for responsiveness to vagusnerve stimulation may include: obtaining a sample of blood taken fromthe patient prior to stimulation of the patient's vagus nerve;challenging the sample of blood with LPS at a first concentration ofLPS; measuring a level of an inflammatory cytokine that is released inresponse to the LPS challenge at the first concentration of LPS;challenging the sample of blood with LPS at a second concentration ofLPS that is at least 5 times the first concentration of LPS; measuring alevel of the inflammatory cytokine that is released in response to theLPS challenge at the second concentration of LPS; and determining aresponse curve based on the level of the inflammatory cytokine that isreleased in response to the LPS challenges at the first concentration ofLPS and the second concentration of LPS; comparing the determinedresponse curve to a cutoff; and determining whether the patient issuitable for vagus nerve stimulation based on the comparison of thedetermined response curve to the cutoff.

The second concentration of LPS may be at least 10 times the firstconcentration of LPS. The method may also include challenging the sampleof blood with LPS at a third concentration of LPS that is at least 5times the second concentration of LPS, and wherein the response curve isdetermined based on the level of the inflammatory cytokine that isreleased in response to the LPS challenges at the first concentration ofLPS, the second concentration of LPS, and the third concentration ofLPS. The inflammatory cytokine may be selected from the group consistingof TNF, IL-1, IL-6, IL-12, IL-18, inflammsome, interferon gamma, andgranulocyte-macrophage colony stimulating factor (e.g., TNF). The stepof determining the response curve may include calculating a slope of theresponse curve. Comparing the determined response curve to the cutoffmay include comparing the slope of the response curve to the cutoff.

Determining whether the patient is suitable for vagus nerve stimulationmay include determining whether the slope of the response curve isgreater than the cutoff.

Also described herein are methods that examine the levels of one or morebiomarker(s) before and after an intentional vagal nerve stimulation(e.g., a mechanical and/or electrical stimulation of the vagus nerve).For example, a method for screening a patient for responsiveness tovagus nerve stimulation may include: measuring a baseline level ofC-reactive protein (CRP) in a sample of blood taken from the patient;intentionally stimulating the vagus nerve (e.g., mechanically and/orelectrically); measuring a post-stimulation level of CRP in a sample ofblood taken after the vagus nerve has been stimulated; comparing thepost-stimulation level of CRP to the baseline level of CRP; andindicating if the patient is a suitable candidate for an implantablevagus nerve stimulation device based on the comparison of thepost-stimulation level of CRP to the baseline level of CRP.

Indicating may comprise indicating that the patient is a suitablecandidate for the implantable vagus nerve stimulation device when thepost-stimulation level of CRP increases by 5% or more.

The vagus nerve may be stimulated in any appropriate manner, includingnoninvasively, with transcutaneous electrical stimulation, by mechanicalstimulation, stimulating the auricular branch of the vagus nerve,stimulating a cervical portion of the vagus nerve, or combinations ofthese. For example, any of these methods may include: introducing anelectrode intravascularly via percutaneous puncture; and positioning theelectrode at a cervically located blood vessel proximate the vagusnerve. In some variations the method may include introducing anelectrode into the carotid sheath; and positioning the electrode withinthe carotid sheath such that the electrode is proximate the vagus nerve.The methods may include: placing an electrode of a transcutaneouselectrical nerve stimulation device on the patient's skin over thecervical vagus nerve.

For example, a method of treating a patient having an inflammatorydisorder with vagus nerve stimulation may include: measuring a baselinelevel of C-reactive protein (CRP) in a sample of blood taken from thepatient; stimulating the patient's vagus nerve; measuring apost-stimulation level of CRP in a sample of blood taken after the vagusnerve has been stimulated; comparing the post-stimulation level of CRPto the baseline level of CRP; and implanting a vagus nerve stimulator ifthe patient is a suitable candidate for an implantable vagus nervestimulation device based on the comparison of the post-stimulation levelof CRP to the baseline level of CRP.

A method for screening a patient for responsiveness to vagus nervestimulation may include: measuring a baseline level of a cytokine andone or more of lymphocytes and monocytes in a sample of blood taken fromthe patient; stimulating the vagus nerve; measuring a post-stimulationlevel of the cytokine and the one or more of lymphocytes and monocytesin a sample of blood taken after the vagus nerve has been stimulated;comparing the post-stimulation level of the cytokine and the one or moreof lymphocytes and monocytes to the baseline level of the cytokine andthe one or more of lymphocytes and monocytes; and indicating if thepatient is a suitable candidate for an implantable vagus nervestimulation device based on the comparison of the post-stimulationlevels of the cytokine and the one or more of lymphocytes and monocytesto the baseline levels of the cytokine and the one or more oflymphocytes and monocytes.

The cytokine may comprise interleukin 7 (IL-7) and the one or more oflymphocytes and monocytes comprise CD3− CD19+ cells (e.g., cellsrecognized by the well-known CD3 antibody recognize some human Tlymphocyte, and CD19 antibody recognizes human B lymphocytes).

Indicating may comprise indicating a patient is a responder if thepatient indicated an increase or a decrease of 10% or less (e.g., 10% orless, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% orless, 3% or less, 2% or less, 1% or less, etc., less than 10%, etc.) inthe cytokine and the one or more of lymphocytes and monocytes comparedto baseline. Indicating may comprise indicating that the patient is anon-responder if the cytokine and the one or more of lymphocytes andmonocytes exhibited a greater than 10% (e.g., greater than 11%, greaterthan 12%, greater than 13%, greater than 14%, greater than 15%, greaterthan 20%, greater than 25%, etc.) decrease in CD3−, CD19+ (e.g.,B-lymphocytes) cell populations and a greater than 10% decrease in IL-7levels compered to baseline. The method vagus nerve may be stimulatednoninvasively, and/or with transcutaneous electrical stimulation, and/orby mechanical stimulation, and/or by stimulating the auricular branch ofthe vagus nerve, and/or by stimulating a cervical portion of the vagusnerve, etc. For example, the method may include introducing an electrodeintravascularly via percutaneous puncture; and positioning the electrodeat a cervically located blood vessel proximate the vagus nerve. Themethod may include introducing an electrode into the carotid sheath; andpositioning the electrode within the carotid sheath such that theelectrode is proximate the vagus nerve. The method may include: placingan electrode of a transcutaneous electrical nerve stimulation device onthe patient's skin over the cervical vagus nerve.

Also described herein are methods of treating a patient having aninflammatory disorder with vagus nerve stimulation, the methodcomprising: measuring a baseline level of a cytokine and one or more oflymphocytes and monocytes in a sample of blood taken from the patient;stimulating the vagus nerve; measuring a post-stimulation level of thecytokine and the one or more of lymphocytes and monocytes in a sample ofblood taken after the vagus nerve has been stimulated; comparing thepost-stimulation level of the cytokine and the one or more oflymphocytes and monocytes to the baseline level of the cytokine and theone or more of lymphocytes and monocytes; and implanting a vagus nervestimulator if the patient is a suitable candidate for an implantablevagus nerve stimulation device based on the comparison of thepost-stimulation level of the cytokine and the one or more oflymphocytes and monocytes compared to the baseline level of the cytokineand the one or more of lymphocytes and monocytes.

For example, described herein are methods for screening a patient forresponsiveness to vagus nerve stimulation comprising all of some of thefollowing steps: obtaining a sample of blood taken from the patientprior to application of vagal nerve stimulation of the patient;challenging the sample of blood with an agent that evokes an immuneresponse, e.g., a toxin or particularly an endotoxin, such asLipopolysaccharides (LPS), at a first concentration; measuring a levelof an inflammatory cytokine that is released in response to thechallenge at the first concentration; challenging the sample of bloodwith the same or a different toxin (and particularly an endotoxin suchas LPS) at a second concentration that is higher than the firstconcentration (e.g., at least twice, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×,11×, 15×, 20×, 25×, 30×, etc.); measuring a level of the inflammatorycytokine that is released in response to the second challenge at thesecond concentration; and determining if the patient is suitable forvagus nerve stimulation based on the response to the second challengerelative to the first challenge. For example, determining if the patientis suitable for vagus nerve stimulation may be based on a response curveof the level of the inflammatory cytokine that is released in responseto the challenges at the first concentration (e.g. of LPS) and thesecond concentration (e.g., of LPS), and comparing the determinedresponse curve to a threshold.

For example, when the challenge is an LPS challenge, the secondconcentration of LPS may be at least 10 times the first concentration ofLPS.

Any of these methods may also include challenging the sample of bloodwith a third concentration of toxin (e.g., endotoxin such as LPS) thatis at twice (e.g., at least 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 11×,12×, 15×, 20×, 25×, 30×, etc.) the second concentration, and wherein thefitness for vagal nerve stimulation is determined based on the level ofthe inflammatory cytokine that is released in response to the thirdchallenges relative to the first and/or second challenge concentrations.

In any of the methods described herein, the inflammatory cytokineexamined from the blood sample may be one or more of: TNF, IL-1, IL-6,IL-12, IL-18, inflammsome, interferon gamma, and granulocyte-macrophagecolony stimulating factor. The inflammatory cytokine may be TNF.

In any of these example, a response curve may be determined. Forexample, determining a response curve may include calculating a slope ofthe response curve. The determined response curve may be compared to acutoff, e.g., by comparing the slope of the response curve to thecutoff.

Determining whether the patient is not suitable for vagus nervestimulation may include determining whether the slope of the responsecurve is less than the cutoff.

In general, the methods described herein may be performed on blood exvivo; the blood sample may be withdrawn from the patient and stored(e.g., frozen, etc.) or used fresh, and challenged over time orsimultaneously with multiple levels of toxin (e.g., endotoxin). This mayallow the patient to be screened without any associated risk to thepatient, prior to any surgical intervention, including implanting aneurostimulator (vagus stimulator).

For example, venous blood may be drawn from the patient, ananticoagulant (e.g., heparin) added, and the blood may be aliquoted totubes. Thereafter a toxin (endotoxin, such as LPS) may be mixed with theblood to final concentrations, e.g., of 0, 1, 10, and 100 ng/mL; theblood may be incubated, e.g., for 4 hr at 37° C. The plasma may beseparated and an immune response (e.g., TNF level) measured, e.g., byELISA.

Also described herein are methods of screening patient based oncomparing a response of an inflammatory marker (e.g., C-reactiveprotein, CRP) before and after vagal nerve stimulation. Vagal nervestimulation (VNS) may be performed anywhere on the vagus nerve, orand/or an associated nucleus. For example, VNS may be performed bytranscranial stimulation to nucleus tractus solitarius (NTS), dorsalmotor nucleus of the vagus (DMV), or Locus coeruleus (LC), for example.

For example, a method for screening a patient for responsiveness tovagus nerve stimulation may include: measuring a baseline level of CRPin a sample of blood taken before the vagus nerve has been stimulated;stimulating the vagus nerve after the step of measuring the baselinelevel of CRP; measuring a post stimulation level of CRP in a sample ofblood taken after the vagus nerve has been stimulated; comparing thepost stimulation level of CRP to the baseline level of CRP; anddetermining whether the patient is a suitable candidate for animplantable vagus nerve stimulation device based on the post-stimulationlevel of CRP relative to the baseline level of CRP. The stimulation ofthe vagus nerve may be done noninvasively. The vagus nerve stimulationmay be done with transcutaneous electrical stimulation. The vagus nervestimulation may be done with mechanical stimulation. The step ofstimulating the vagus nerve may comprise stimulating the auricularbranch of the vagus nerve. The stimulation of the vagus nerve maycomprise stimulating a cervical portion of the vagus nerve.

For example, the method may include introducing an electrodeintravascularly via percutaneous puncture; and positioning the electrodeat a cervically located blood vessel proximate the vagus nerve.

The method may include introducing an electrode into the carotid sheath;and positioning the electrode within the carotid sheath such that theelectrode is proximate the vagus nerve. Alternatively or additionally,the method may include placing an electrode of a transcutaneouselectrical nerve stimulation device on the patient's skin over thecervical vagus nerve.

Any of the pre-screening methods described herein may also include oneor more of: administration of nicotinic agonist, for example, anicotinic a7 agonists, and/or one or more cholinesterase inhibitor(s).

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1C illustrate various embodiments of a mechanical stimulator.

FIG. 2 illustrates an embodiment of a transcutaneous electrical nervestimulation device.

FIGS. 3A-3C illustrate various embodiments of minimally invasiveelectrical stimulation of the vagus nerve.

FIG. 3D illustrates an embodiment of minimally invasive mechanicalstimulation of the vagus nerve.

FIGS. 4A-4C illustrate that that there was no obvious or significantdifference in LPS-induced TNF levels under various LPS concentrationsbetween European League Against Rheumatism (EULAR) criteria respondersand nonresponders in the two cohorts of patients tested.

FIG. 5 illustrates LPS dose response curves for responders andnonresponders using blood samples before vagus nerve stimulation.

FIG. 6 illustrates LPS dose response curves for responders andnonresponders using blood samples before vagus nerve stimulation.

FIG. 7 illustrates the slopes of the LPS dose response curves forresponders and non-responders to 100 ng/ml LPS.

FIG. 8 illustrate the slopes of the LPS dose response curves forresponders and non-responders to 10 ng/ml LPS.

FIG. 9 illustrates the change in CRP levels after vagus nervestimulation comparing responders and nonresponders.

FIG. 10 illustrates the change in CRP levels after vagus nervestimulation for responders and nonresponders (based on the change frombaseline).

FIG. 11 illustrates another example of the change in CRP levels aftervagus nerve stimulation for responders and nonresponders (based on thechange from baseline).

FIG. 12 is a table that shows changes in cell populations and IL-7levels between screening day and after implantation of the vagus nervestimulation device.

FIG. 13 is a table that shows changes in cell populations and IL-7levels between screening day and after implantation of the vagus nervestimulation device.

DETAILED DESCRIPTION

In general, described herein are methods and apparatuses fordetermining, e.g., by a blood test, if a subject (e.g., a mammalian,including human, patient) will benefit and/or respond to electricaland/or mechanical vagus nerve stimulation (VNS) in order to treat aninflammatory disorder.

In particular, described herein are methods and apparatuses (devices,systems, assays, etc.) for taking a blood sample from a patient andperforming one or more procedures on the blood sample, without having tosubject the patient to any stimulation. This may allow for theidentification of patients that may benefit from the therapy (or mayrequire additional or adjunctive therapies or modifications in the levelof therapy provided) prior to providing any therapy to the patient.Since the methods may be performed just on an ex vivo sample removedfrom the patient's body, the patient may be spared any risk,complications, and/or discomfort due to stimulation.

Described herein are three variations of the ex vivo methods fordetermining if a patient will respond to vagus nerve stimulation. Forexample, described herein are methods for treatment of RheumatoidArthritis (RA) by activating the CAP using electrically activeimplantable medical devices; these methods may include first confirmingthat the patient is a candidate for treatment using any of the methodsdescribed herein.

For example, a study of vagus nerve stimulation (VNS) using anelectrically active surgically implanted medical device in 8 patientswith active rheumatoid arthritis (RA) was performed at 4 investigativecenters. The device used an implanted helical coiled cuff lead todeliver electrical stimulation from an implanted pulse generator. After6 weeks of stimulation, clinically significant improvements were seen in6 of 8 patients as assessed by standard RA efficacy outcome measuressuch as the Disease Activity Score (DAS). These results provideproof-of-concept for the therapeutic use of VNS in RA as an alternativeto small molecule and biological agent therapy. However, while themajority of the patients in the study responded, 2 of 8 did not have ameaningful clinical response. In order for VNS to be more successful asa therapy, it would be desirable to use a diagnostic screening test topreoperatively predict likelihood of clinical response to an implant,thus sparing patients who are unlikely to respond from undergoing anunnecessary surgical procedure. The diagnostic screening tests describedherein may utilize various techniques, such as mechanical and electricalstimulation to stimulate the vagus nerve noninvasively and/or in somecases an iv vitro toxin challenge, to determine if a patient willrespond to treatment prior to implantation of the electricalstimulation.

For example, in some variations, the effectiveness of CAP Activation maybe determined by stimulation of the auricular branch of the vagus verveprior to implantation. Sensory fibers of the Auricular Branch of theVagus Nerve (ABVN) innervate the skin of the cymba concha of theexternal ear. These fibers may provide afferent input via the superiorganglion of the vagus to the brainstem nucleus of the solitary tract(NST). The neurons of the NST then project to efferent neuronsoriginating in the dorsal nucleus and nucleus ambiguous of the vagusnerve, which then in turn project within the vagus nerve to the visceralorgans.

This neuroanatomical pathway may provide a potential mechanism fornon-invasive activation of the CAP by induction of efferent vagaloutflow through stimulation of the afferent ABVN pathway usingmechanical or electrical stimulation of the skin of the cymba concha.The ABVN is particularly suitable for noninvasive stimulation becausethe nerves are located relatively close to the surface of the skin.Similarly, other portions of the vagus nerve that are similarly situatedclose to the patient's skin may be used for noninvasive stimulation. Forexample, the nerve may be located less than about 2, 1, 0.5 or 0.25 cmfrom the surface of the patient's skin. The terms “about”,“approximately” and the like can mean within 10%, 20%, or 30%.

The presence of such a functional reflex pathway (immune reflex pathway)in the ABVN is suggested by the following observations: (1) the“Arnold's reflex” occurs in approximately 2% of the population andresults in coughing or gagging in response to mechanical stimulation ofthe ear canal; (2) ABVN electrical stimulation in rats resulted inparasympathetic vagally-mediated reductions in heart rate and bloodpressure, and increased intra-gastric pressure. This response could beabrogated with atropine; (3) ABVN electrical stimulation in dogsreverses the acute right atrial remodeling and reduction in thresholdfor inducible atrial fibrillation caused by rapid right atrial pacing;and this protective effect of ABVN could be eliminated by bilateraltransection of the vagus in its upper thoracic segment; (4) the abilityof ABVN stimulation to activate the CAP and inhibit systemicinflammation in rodents. In a rodent systemic endotoxemia model,transcutaneous electrical ABVN stimulation was compared to VNS deliveredusing a surgically placed cervical electrode. While cervical VNS was themost effective, ABVN stimulation caused significant reductions incirculating tumor necrosis factor (TNF), Interleukin (IL)-1 beta, andIL-6, thus demonstrating that the CAP can be effectively activated bytranscutaneous auricular stimulation.

Described herein are methods and apparatuses for using the CAPactivation as a diagnostic screening test by applying a short durationstimulation of the skin of the external ear, for example eithermechanically or with an electric current. The stimulation duration canbe between about 1 second to 24 hours and can be applied eithercontinuously or intermittently throughout the duration. In someembodiments, the duration is less than about 1, 5 10, 20, 30, or 60seconds. In some embodiments, the duration is less than about 1, 2, 3,4, 5, 10, 15, 20, 30, 40, 50, or 60 minutes. In some embodiments, theduration is less than about 1, 2, 3, 4, 5, 6, 12, 18, or 24 hours.

The non-invasive stimulation may include mechanical stimulation of abody region such as the subject's ear. Examples of non-invasivestimulation devices are described in U.S. Publication No. 2008/0249439to Tracey et al., which is herein incorporated by reference in itsentirety. In particular, the cymba conchae region of their ear may bestimulated. The non-invasive stimulation may comprise mechanicalstimulation between about 1 and 500 Hz, or 30 Hz and 500 Hz, or 50 Hzand 500 Hz. In some variations the stimulation is transcutaneousstimulation applied to the appropriate body region (e.g., the ear). Forexample, transcutaneous stimulation may be applied for an appropriateduration (e.g., less than 24 hours to less than 1 hour, less than 60minutes to less than 1 minute, less than 60 seconds to less than 1second, etc.), at an appropriate intensity and frequency. Stimulationthat does not significantly affect cardiac measures may be particularlydesirable, and the stimulation may be limited to such a range, or may beregulated by cardiac feedback (e.g., ECG, etc.).

Also described herein are devices for non-invasively mechanicallystimulating a subject's inflammatory reflex, as illustrated in FIGS.1A-1C. These mechanical stimulation devices 100 may include an actuator102, such as a movable distal tip region that is configured tomechanically stimulate at least a portion of a subject's ear, a handle104, and a driver 106 configured to move the distal tip region betweenabout 50 and 500 Hz. In some variations, the stimulation devices arepart of a system including a stimulation device. In some embodiments,the actuator 102 can be directly adhered to the patient's skin, therebyremoving the need for a handle. The actuator 102 can be coated with anadhesive or can be integrated into an adhesive pad or pod 108.

A stimulation device may include a controller configured to control thedriver so that it applies stimulation within stimulation parameters. Forexample the controller (which may be part of the driver, or may beseparate from the driver) may control the intensity (e.g., force,displacement, etc.), the timing and/or frequency (e.g., the frequency ofrepeated pulses during a stimulation period, the stimulation durationduring the period of stimulation, the duration between stimulationperiods, etc.), or the like. In some variations the controller ispre-programmed. In some variations, the controller receives input. Theinput may be control input (e.g., from a physician or the patient) thatmodifies the treatment. In some variations the stimulator deviceincludes a therapy timer configured to limit the duration ofstimulation. For example, the controller may be configured to limit theperiod of stimulation to less than 10 minutes, less than 5 minutes, lessthan 3 minutes, less than 1 minute, etc.

Any appropriate driver may be used. For example, the driver may be amotor, voice (or speaker) coil, electromagnet, bimorph, piezo crystal,electrostatic actuator, and/or rotating magnet or mass. For example, insome variations the driver is a mechanical driver that moves an actuatoragainst the subject's skin. Thus, an actuator may be a distal tip regionhaving a diameter of between about 35 mm and about 8 mm.

In some variation the stimulator includes a frequency generator that isin communication with the driver. Thus the driver may control thefrequency generator to apply a particular predetermined frequency orrange of frequencies to the actuator to non-invasively stimulate thesubject.

The stimulator devices described herein may be hand-held or wearable.For example, also described herein are wearable device fornon-invasively stimulating a subject's inflammatory reflex. Thesestimulator the devices may include an actuator configured tomechanically stimulate a subject's cymba conchae, a driver configured tomove the distal tip region between about 1 and 500 Hz, or 30 Hz and 500Hz, or 50 Hz and 500 Hz, and an ear attachment region configured tosecure to at least a portion of a subject's ear.

Transcutaneous electrical nerve stimulation (TENS) can be provided by anelectrical stimulation device 200 having at least one electrode 202 thatcan be placed on the patient's skin, as illustrated in FIG. 2 . Theelectrode 202 can be integrated into an adhesive patch or pad 204 thatcan be adhered to the patient's skin. A lead can connect the electrodewith the housing 206 of the device. Alternatively, the housing can alsobe integrated with the adhesive patch or pad. A control or signalgenerator can deliver the electrical signal stimulus through theelectrode. The signal amplitude can be between about 0.05 to 10 mA, or0.05 to 15 mA, or 0.05 to 20 mA, or 0.05 to 25 mA, or 0.05 to 50 mA. Thepulse width can be between about 100 and 1,000 μS. The pulse frequencycan be between about 1 and 50 Hz, and the stimulus duration can bebetween about 1 second and 24 hours.

The electrical parameters can be similar when the electrical stimulationis provided by an electrode that has been inserted into the patientthrough minimally invasive techniques as described herein. For example,the signal amplitude can be between about 0.05 to 5 mA, or 0.05 to 10mA, or 0.05 to 15 mA, 0.05 to 20 mA, or 0.05 to 25 mA or 0.05 to 50 mA.

In some variations a method for treating a patient and/or determining ifa patient is a candidate for treatment may include CAP activation bystimulation of the cervical vagus nerve. These methods may includeacutely invasive techniques. For example, temporary electricalstimulation of the cervical vagus nerve for screening purposes can beperformed using a variety of devices. A device can include a non-cuffedlead 306 placed percutaneously through a blood vessel 304 such as theinternal jugular vein on or near the vagus nerve 302 within the carotidsheath 300, as illustrated in FIG. 3A. Alternatively, as shown in FIG.3B, a standard percutaneous catheter-directed intravascular electrode308 can be positioned within the internal jugular 304 or other nearbyblood vessel, in close anatomical apposition to the vagus nerve 302,with electrical stimulation delivered trans-vascularly. Other electrodeor lead configurations, such as needle electrodes 310, can also be usedto temporarily stimulate the vagus nerve 302, as shown in FIG. 3C.

Alternatively, direct mechanical stimulation of the cervical vagus nerveby physical manipulation has been demonstrated to activate the CAP inthe rodent endotoxemia model. Angioplasty of the internal jugular veinmay be performed using percutaneously inserted balloon-tippedangioplasty catheters. Transmural pressure can be exerted internallywithin the internal jugular vein 304 using such balloon catheters 312 inorder to mechanically stimulate the nearby cervical vagus nerve 302 andactivate the CAP, as shown in FIG. 3D. Other mechanical stimulators orexpanders can also be used to provide mechanical stimulation.

Alternatively, the cervical vagus nerve can also be stimulated usingintravascular or transcutaneous ultrasound, transcutaneous magneticenergy, transcutaneous RF energy, and transcutaneous electricstimulation.

The CAP can be activated in the diagnostic screening test describedherein by short duration (1 second to 24 hour) stimulation of thecervical vagus nerve using the above methods, either mechanically orwith an electric current.

Furthermore, in some variations, CAP activation specific to a patientmay be examined using a bioassay performed on one or more peripheralblood samples. The degree of CAP activation induced by the stimulationmethods described above can be measured in a diagnostic screening testusing several biological activity assays performed on either serumsamples, supernatants from whole blood samples cultured in vitro in thepresence of cytokine release stimulators, or supernatants from separatedcellular constituents of whole blood cultured in vitro in the presenceof cytokine release stimulators. Alternatively or additionally, theassay may be based on tissue samples or other biological fluids. Thereduction or increase in these mediators that is observed between apre-stimulus measurement and a post-stimulus measurement, and/or betweenstimulation at different levels, may be indicative of the responsivenessof the patient to CAP activation, and may predict the response to apermanently implanted VNS system. For example, a predetermined change inlevel or concentration of a mediator, cytokine, or analyte from abaseline level or concentration before stimulation may indicateresponsiveness of the CAP to stimulation and suitability of the patientfor an implanted VNS system. Alternatively, the reduction or increase ininducible release of a cytokine, mediator, signaling molecule, or otheranalyte in a cell-based assay can also be used. In some embodiments, thepredetermined change is a reduction of at least 10, 20, 30, 40 or 50%.In some embodiments, the predetermined change is an increase of at least10, 20, 30, 40 or 50%.

For example, one bioassay measures endotoxin, e.g., lipopolysaccharide(LPS), induced TNF levels in whole blood samples taken during thescreening day before implantation of the VNS stimulator or before anystimulation of the vagus nerve. FIGS. 4A-8 illustrate one set ofexperiments characterizing an in vitro assay in which blood samples fromboth responders and non-responders (taken before implantation) werecompared. To perform the whole blood assay (WBA), venous blood was drawnfrom each patient and dispensed with heparin into separate tubes on ascreening day (e.g., day −21). LPS (endotoxin) was mixed with the bloodto final concentrations of 0, 1, 10, and 100 ng/mL. Blood was incubatedfor 4 hours at 37° Celsius. Plasma was then separated from the wholeblood and TNF levels were measured by ELISA. On Day −14, seven daysafter the screening day, the patients were then implanted with acervical vagus nerve stimulation device for treatment of inflammationcaused by rheumatoid arthritis, and on Day 0, two weeks afterimplantation the vagus nerve was stimulated with the implanted deviceand subsequently stimulated according to a prescribed therapy regimen.The patients were characterized as responders or nonresponders based ontheir response to the VNS therapy. The screening test data was thenanalyzed.

FIGS. 4A-4C illustrate that there was no obvious or significantdifference in LPS-induced TNF levels under various LPS concentrationsbetween European League Against Rheumatism (EULAR) criteria respondersand nonresponders in the two cohorts of patients tested. No differenceis seen because the amount of TNF produced appears to be highly patientspecific.

However, a response curve analysis of the TNF level data surprisinglywas able to identify and/or separate four out of five nonresponders fromthe two cohorts of patients (18 patients total—5 nonresponders and 13responders) with a high level of significance. To perform the responsecurve analysis, patient-specific TNF levels produced at various LPSconcentrations were normalized to TNF levels at 1 ng/mL LPS. NormalizedTNF levels produced as a function of LPS concentration was plotted byindividual and EULAR response, as shown in FIG. 5 , and by cohort andEULAR response, as shown in FIG. 6 . Slopes of the response curves werecalculated, grouped, and plotted, as shown in FIGS. 7 and 8 . For twopatients, there was undetectable levels of TNF at 1 ng/mL LPS, so theslopes were extrapolated from the change between 10 and 100 ng/mL LPS.

Based on the data presented in FIG. 7 which presents TNF response dataover a 3 log LPS concentration range (1 to 100 ng/mL LPS challenge), 4of the 5 nonresponders had a slope of less than about 60 while all butone responder had a slope greater than about 60. Similarly, FIG. 8presents TNF response data over a 2 log LPS concentration range (1 to 10ng/mL LPS challenge), where 4 out of 5 nonresponders had a slope lessthan about 75 while all but two responders had a slope greater thanabout 75.

FIGS. 7 and 8 demonstrate that a screening test based on an assay thatmeasures TNF release or release of another inflammatory cytokine, suchas IL-1, IL-6, IL-12, IL-18, inflammsome, interferon gamma, andgranulocyte-macrophage colony stimulating factor, from blood cells,particularly white blood cells, in response to LPS/endotoxin challengeover a range of LPS/endotoxin concentrations, can be used to effectivelyscreen patients before implantation to determine whether a candidate islikely to respond or not respond to VNS therapy. In other words, anassay, such as a whole blood assay, that measures a response curve orresponse over range of challenge concentrations can be used as ascreening test. In some embodiments, the range of concentration for theLPS/endotoxin can be at least 2, 3, or 4 logs, where 2 logs means thatthe highest concentration is 10 times the lowest concentration, and 3longs means that the highest concentration is 100 times the lowestconcentration. In some embodiments, the concentrations may be increasedin multiples of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Thescreening test can have a cutoff value for the response curve, such asthe slope of the response curve or another measure of the degree ormagnitude of the response over the concentration range, that establisheswhether a candidate is suitable for VNS therapy. In some embodiments,the WBA can be performed using a commercial blood testing kit, such as aTruCulture® kit sold by Myriad RBM. In some embodiments, the blood canbe incubated for less than 4 hours, such as less than 3, 2, or 1 hour,or incubation can be greater than 1, 2, 3, or 4 hours. In someembodiments, the assay can use whole blood, or it can use isolated whiteblood cells or monocytes. In some embodiments, the cells can bestimulated by LPS/endotoxin, while in other embodiments, other stimulican be used to stimulate the blood cells. In some embodiments, theLPS/endotoxin concentration can range from about 1 ng/mL to about 100ng/mL. In other embodiments, the low end for the LPS/endotoxinconcentration can be less than 1 ng/mL, such as about 100 pg/mL or 10pg/mL, and the high end can be about 1000 ng/mL or 10000 ng/mL.

Other tests may be performed after stimulation of the vagus nerve usingnoninvasive or minimally invasive devices. For example, C-reactiveprotein (CRP) is a protein synthesized in the liver and secreted intothe blood in response to inflammation, particularly in response to IL-6secretion by macrophages and T-cells. The prospective patient's bloodcan be collected during the screening day and again after the patient'svagus nerve has been stimulated noninvasively or minimally invasively.After a set or predetermined amount of time after VNS, such as 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, the second blood samplecan be collected and both samples can be tested for a biomarker, such asCRP. The first sample taken before VNS stimulation can establish abaseline or reference level of the analyte of interest, and the secondblood sample can establish the effect of VNS stimulation on thebiomarker. For patients that are likely to respond well to VNS therapy,the CRP levels are likely to remain stable or be reduced, while patientsthat do not respond well to VNS therapy may show elevated levels of CRP,which may indicate worsening of inflammation. Therefore, patients thatdo not respond well to VNS therapy may be identified and excluded bysetting a cutoff or threshold value based on a comparison of the CRPlevels in the second sample to the reference CRP level, as shown inFIGS. 9-11 , which show the change in CRP levels at Day 7 (seven daysafter initial VNS stimulation) with respect to a baseline level forresponders and nonresponders (identified at Day 42 using EULARcriteria). For example, the data shown in FIGS. 9-11 indicate that theCRP levels of all the nonresponders increased by at least about 5 to 10percent relative to the reference CRP level. Therefore, the cutoff forexcluding patients can be an increase in CRP levels of greater than 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 percent after VNS (e.g.,non-invasive VNS or acutely invasive VNS).

FIGS. 12 and 13 present changes in cell populations and IL-7 levels fromD-21 (screening day) to D0 (14 days after implantation of VNS device butbefore delivering any stimulation). All nonresponders exhibited greaterthan 10% decrease in CD3-CD19+(B-lymphocytes) cell populations andgreater than 10% decrease in IL-7 levels (see FIG. 13 ).

Thus a method of treating a patient may include determining if thepatient will respond to the treatment based on the relationship of oneor more monocyte and lymphocyte populations after a transcutaneous vagusnerve stimulation during screening, particularly in combination with abiomarker such as an inflammatory marker (e.g., IL-7) by comparing thelevel of the monocytes and/or lymphocytes and the level of theinflammatory biomarker from a baseline taken prior to any treatment witha level following a non-invasive or acutely invasive treatment. Inparticular, if the level of the monocytes and/or lymphocytes (e.g.,CD3−, CD19+ B-lymphocytes) and the level of the cytokine (e.g., IL-7)both decrease, the patient may be a non-responder, and implantation maybe avoided. In patients for whom either or both the level of monocytesand/or lymphocytes (e.g., CD3−, CD19+ B lymphocytes) and the level ofthe cytokine (e.g., IL-7) increase between baseline and non-invasive oracutely invasive stimulation (which may be performed immediately priorto implantation), implantation may be completed as they are likely torespond to treatment.

Diagnostic Screening Tests

A diagnostic screening test can predict the likelihood of clinicalresponse to the VNS implant prior to actual implantation surgery (orcompletion of implantation), for use in clinical decision-makingregarding patient selection. Patients that are in need of or that maybenefit from a VNS implant can be identified and given the diagnosticscreening test. In some embodiments, the test is performed prior toimplantation of the device and/or any invasive or noninvasivestimulation of the vagus nerve. In other embodiments, the test involvesactivating the cholinergic anti-inflammatory pathway for a shortduration using techniques that are either non-invasive or minimallyinvasive as described herein. For example, the vagus nerve can bestimulated electrically or mechanically, as described herein. The extentof the patient's biological response to temporary CAP activation maythen be assessed by measuring pathway-mediated inhibition of immuneactivation in assays performed on blood samples or other biologicalfluids taken before and after the stimulation. Surprisingly, the extentof the patient's biological response to a brief non- or minimallyinvasive (e.g., acutely invasive) activation of the pathway can predictclinical response to the permanent implant.

Activation of the cholinergic anti-inflammatory pathway may be achievedthrough any of the following stimulation techniques: (1) noninvasivetranscutaneous stimulation of the auricular branch of the vagus nervewhich innervates the skin of the cymba conchae of the ear, using eitherelectrical or mechanical stimulation; (2) stimulation of the cervicalvagus nerve using a catheter-directed temporary electricallead/electrode, introduced intravascularly via percutaneous puncture,and positioned within the cervical internal jugular vein or other nearbyvein under fluoroscopic or ultrasound guidance to place it in closeapposition to the cervical portion of the vagus nerve; (3) stimulationof the cervical vagus nerve by a temporary catheter-directed non-cuffedelectrical lead/electrode, introduced into the carotid sheath bypercutaneous puncture, and positioned under fluoroscopic, ultrasound,and/or endoscopic guidance to place it in close apposition to thecervical portion of the vagus nerve within the carotid sheath; (4)stimulation of the cervical vagus nerve by trans-vascular mechanicalpressure using an intravascular angioplasty balloon or other expandableelement, introduced via percutaneous puncture and positioned underfluoroscopic or ultrasound guidance in close apposition to the cervicalportion of the vagus nerve within the cervical internal jugular vein orother nearby vein; and (5) noninvasive transcutaneous cervical vagusnerve stimulation using a transcutaneous electrical nerve stimulationdevice placed over the vagus nerve on the skin of the patient's neck.

Assessment of the strength of CAP activation can measured using thechange from pre- to post stimulation levels in any of the followingparameters in a tissue sample or biological fluid such as whole blood,serum, or supernatants from cultures of whole blood, or from cellsisolated from whole blood: (1) cytokines or inflammatory mediators orother analytes; and (2) reduction in inducible release of cytokines orother inflammatory mediators or other analytes, from in vitro culture ofwhole blood or isolated cells from blood. Such induction may be in theform of bacterial lipopolysaccharide, other Toll-like receptoractivators, fragments of complement molecules (e.g., C5a), or activatingantibodies directed against T cell or B cell surface receptors (e.g.anti CD3/anti CD28 antibodies).

Alternative Stimulation Locations

Other regions of the subject's body may be alternatively or additionallystimulated, particularly regions enervated by nerves of the inflammatoryreflex. For example, the non-invasive stimulation and other stimulationmodalities described herein may be applied to the subject's areainnervated by the seventh (facial) cranial nerve or cranial nerve V. Thenon-invasive stimulation and other stimulation modalities describedherein may be applied to at least one location selected from: thesubject's cymba conchae of the ear, or helix of the ear. In somevariations, the non-invasive stimulation and other stimulationmodalities described herein is applied to at least one point along thespleen meridian.

It is understood that this disclosure, in many respects, is onlyillustrative of the numerous alternative device embodiments of thepresent invention. Changes may be made in the details, particularly inmatters of shape, size, material and arrangement of various devicecomponents without exceeding the scope of the various embodiments of theinvention. Those skilled in the art will appreciate that the exemplaryembodiments and descriptions thereof are merely illustrative of theinvention as a whole. While several principles of the invention are madeclear in the exemplary embodiments described above, those skilled in theart will appreciate that modifications of the structure, arrangement,proportions, elements, materials and methods of use, may be utilized inthe practice of the invention, and otherwise, which are particularlyadapted to specific environments and operative requirements withoutdeparting from the scope of the invention. In addition, while certainfeatures and elements have been described in connection with particularembodiments, those skilled in the art will appreciate that thosefeatures and elements can be combined with the other embodimentsdisclosed herein.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims. The examples and illustrations included herein show, by wayof illustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. An in vitro method for screening a patient forresponsiveness to vagus nerve stimulation to treat an inflammatorydisorder, the method comprising: obtaining a sample of blood taken fromthe patient prior to stimulation of the patient's vagus nerve;challenging the sample of blood with a first concentration of toxin;obtaining a second sample of blood taken from the patient prior tostimulation of the patient's vagus nerve; challenging a second sample ofblood with a second concentration of toxin that is at least 2 times thefirst concentration; determining an amount of inflammatory cytokinereleased in response to the second concentration of toxin compared tothe inflammatory cytokine released in response to the firstconcentration of toxin; and outputting an indication that the patient isa responder or a non-responder for vagus nerve stimulation based on theamount of inflammatory cytokine released.
 2. The method of claim 1,further comprising determining if the patient is a responder for vagusnerve stimulation by determining that a ratio of the amount of theinflammatory cytokine released in response to the second concentrationof toxin and the amount of the inflammatory cytokine released inresponse to the first concentration of toxin is greater than a thresholdratio.
 3. The method of claim 2, wherein the threshold ratio is greaterthan
 60. 4. The method of claim 1, further comprising challenging thesample of blood with the toxin at a third concentration that is at least2 times the second concentration and measuring a level of theinflammatory cytokine that is released in response to the thirdconcentration, wherein outputting the indication that the patient is aresponder for vagus nerve stimulation treatment to treat an inflammatorydisorder is based on the amount of inflammatory cytokine released inresponse to the third concentration of toxin relative to the response tothe first and second concentrations of toxin.
 5. The method of claim 1,wherein challenging the sample of blood with the toxin at a secondconcentration comprises challenging with a concentration that is atleast 10 times the first concentration.
 6. An in vitro method forscreening a patient for responsiveness to vagus nerve stimulation, themethod comprising: obtaining a sample of blood taken from the patientprior to stimulation of the patient's vagus nerve; challenging thesample of blood with LPS at a first concentration of LPS; measuring alevel of an inflammatory cytokine that is released in response to theLPS challenge at the first concentration of LPS; challenging the sampleof blood with LPS at a second concentration of LPS that is at least 5times the first concentration of LPS; measuring a level of theinflammatory cytokine that is released in response to the LPS challengeat the second concentration of LPS; and determining a response curvebased on the level of the inflammatory cytokine that is released inresponse to the LPS challenges at the first concentration of LPS and thesecond concentration of LPS; comparing the determined response curve toa cutoff; and determining whether the patient is suitable for vagusnerve stimulation based on the comparison of the determined responsecurve to the cutoff.
 7. The method of claim 6, wherein the secondconcentration of LPS is at least 10 times the first concentration ofLPS.
 8. The method of claim 6, further comprising challenging the sampleof blood with LPS at a third concentration of LPS that is at least 5times the second concentration of LPS, and wherein the response curve isdetermined based on the level of the inflammatory cytokine that isreleased in response to the LPS challenges at the first concentration ofLPS, the second concentration of LPS, and the third concentration ofLPS.
 9. The method of claim 6, wherein the inflammatory cytokine isselected from the group consisting of TNF, IL-1, IL-6, IL-12, IL-18,inflammsome, interferon gamma, and granulocyte-macrophage colonystimulating factor.
 10. The method of claim 6, wherein the inflammatorycytokine is TNF.
 11. The method of claim 6, wherein the step ofdetermining the response curve includes calculating a slope of theresponse curve.
 12. The method of claim 6, wherein the step of comparingthe determined response curve to the cutoff includes comparing the slopeof the response curve to the cutoff.
 13. The method of claim 12, whereinthe step of determining whether the patient is suitable for vagus nervestimulation includes determining whether the slope of the response curveis greater than the cutoff.
 14. A method for screening a patient forresponsiveness to vagus nerve stimulation, the method comprising:measuring a baseline level of C-reactive protein (CRP) in a sample ofblood taken from the patient; stimulating the vagus nerve; measuring apost-stimulation level of CRP in a sample of blood taken after the vagusnerve has been stimulated; comparing the post-stimulation level of CRPto the baseline level of CRP; and indicating if the patient is asuitable candidate for an implantable vagus nerve stimulation devicebased on the comparison of the post-stimulation level of CRP to thebaseline level of CRP.
 15. The method of claim 14, wherein indicatingcomprises indicating that the patient is a suitable candidate for theimplantable vagus nerve stimulation device when the post-stimulationlevel of CRP increases by 5% or more.
 16. The method of claim 14,wherein the vagus nerve is stimulated noninvasively.
 17. The method ofclaim 14, wherein the vagus nerve is stimulated with transcutaneouselectrical stimulation.
 18. The method of claim 14, wherein the vagusnerve is stimulated by mechanical stimulation.
 19. The method of claim14, wherein the stimulating the vagus nerve comprises stimulating theauricular branch of the vagus nerve.
 20. The method of claim 14, whereinstimulating the vagus nerve comprises stimulating a cervical portion ofthe vagus nerve.
 21. The method of claim 14, further comprising:introducing an electrode intravascularly via percutaneous puncture; andpositioning the electrode at a cervically located blood vessel proximatethe vagus nerve.
 22. The method of claim 14, further comprising:introducing an electrode into the carotid sheath; and positioning theelectrode within the carotid sheath such that the electrode is proximatethe vagus nerve.
 23. The method of claim 14, further comprising: placingan electrode of a transcutaneous electrical nerve stimulation device onthe patient's skin over the cervical vagus nerve.